Quaternary dynamics of the SecA motor drive translocase catalysis.

Most secretory preproteins exit bacterial cells through the protein translocase, comprising the SecYEG channel and the dimeric peripheral ATPase motor SecA. Energetic coupling to work remains elusive. We now demonstrate that translocation is driven by unusually dynamic quaternary changes in SecA. The dimer occupies several successive states with distinct protomer arrangements. SecA docks on SecYEG as a dimer and becomes functionally asymmetric. Docking occurs via only one protomer. The second protomer allosterically regulates downstream steps. Binding of one preprotein signal peptide to the SecYEG-docked SecA protomer elongates the SecA dimer and triggers the translocase holoenzyme to obtain a lower activation energy conformation. ATP hydrolysis monomerizes the triggered SecA dimer, causing mature chain trapping and processive translocation. This is a unique example of one protein exploiting quaternary dynamics to become a substrate receptor, a "loading clamp," and a "processive motor." This mechanism has widespread implications on protein translocases, chaperones, and motors.

Rapid label-free quantitative analysis of the E. coli BL21(DE3) inner membrane proteome.

Biological membranes define cells and cellular compartments and are essential in regulating bidirectional flow of chemicals and signals. Characterizing their protein content therefore is required to determine their function, nevertheless, the comprehensive determination of membrane-embedded sub-proteomes remains challenging. Here, we experimentally characterized the inner membrane proteome (IMP) of the model organism E. coli BL21(DE3). We took advantage of the recent extensive re-annotation of the theoretical E. coli IMP regarding the sub-cellular localization of all its proteins. Using surface proteolysis of IMVs with variable chemical treatments followed by nanoLC-MS/MS analysis, we experimentally identified ∼45% of the expressed IMP in wild type E. coli BL21(DE3) with 242 proteins reported here for the first time. Using modified label-free approaches we quantified 220 IM proteins. Finally, we compared protein levels between wild type cells and those over-synthesizing the membrane-embedded translocation channel SecYEG proteins. We propose that this proteomics pipeline will be generally applicable to the determination of IMP from other bacteria.

SecA-mediated targeting and translocation of secretory proteins.

More than 30 years of research have revealed that the dynamic nanomotor SecA is a central player in bacterial protein secretion. SecA associates with the SecYEG channel and transports polypeptides post-translationally to the trans side of the cytoplasmic membrane. It comprises a helicase-like ATPase core coupled to two domains that provide specificity for preprotein translocation. Apart from SecYEG, SecA associates with multiple ligands like ribosomes, nucleotides, lipids, chaperones and preproteins. It exerts its essential contribution in two phases. First, SecA, alone or in concert with chaperones, helps mediate the targeting of the secretory proteins from the ribosome to the membrane. Next, at the membrane it converts chemical energy to mechanical work and translocates preproteins through the SecYEG channel. SecA is a highly dynamic enzyme, it exploits disorder-order kinetics, swiveling and dissociation of domains and dimer to monomer transformations that are tightly coupled with its catalytic function. Preprotein signal sequences and mature domains exploit these dynamics to manipulate the nanomotor and thus achieve their export at the expense of metabolic energy. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.

The Escherichia coli peripheral inner membrane proteome.

Biological membranes are essential for cell viability. Their functional characteristics strongly depend on their protein content, which consists of transmembrane (integral) and peripherally associated membrane proteins. Both integral and peripheral inner membrane proteins mediate a plethora of biological processes. Whereas transmembrane proteins have characteristic hydrophobic stretches and can be predicted using bioinformatics approaches, peripheral inner membrane proteins are hydrophilic, exist in equilibria with soluble pools, and carry no discernible membrane targeting signals. We experimentally determined the cytoplasmic peripheral inner membrane proteome of the model organism Escherichia coli using a multidisciplinary approach. Initially, we extensively re-annotated the theoretical proteome regarding subcellular localization using literature searches, manual curation, and multi-combinatorial bioinformatics searches of the available databases. Next we used sequential biochemical fractionations coupled to direct identification of individual proteins and protein complexes using high resolution mass spectrometry. We determined that the proposed cytoplasmic peripheral inner membrane proteome occupies a previously unsuspected ∼19% of the basic E. coli BL21(DE3) proteome, and the detected peripheral inner membrane proteome occupies ∼25% of the estimated expressed proteome of this cell grown in LB medium to mid-log phase. This value might increase when fleeting interactions, not studied here, are taken into account. Several proteins previously regarded as exclusively cytoplasmic bind membranes avidly. Many of these proteins are organized in functional or/and structural oligomeric complexes that bind to the membrane with multiple interactions. Identified proteins cover the full spectrum of biological activities, and more than half of them are essential. Our data suggest that the cytoplasmic proteome displays remarkably dynamic and extensive communication with biological membrane surfaces that we are only beginning to decipher.

Preproteins couple the intrinsic dynamics of SecA to its ATPase cycle to translocate via a catch and release mechanism.

Protein machines undergo conformational motions to interact with and manipulate polymeric substrates. The Sec translocase promiscuously recognizes, becomes activated, and secretes >500 non-folded preprotein clients across bacterial cytoplasmic membranes. Here, we reveal that the intrinsic dynamics of the translocase ATPase, SecA, and of preproteins combine to achieve translocation. SecA possesses an intrinsically dynamic preprotein clamp attached to an equally dynamic ATPase motor. Alternating motor conformations are finely controlled by the γ-phosphate of ATP, while ADP causes motor stalling, independently of clamp motions. Functional preproteins physically bridge these independent dynamics. Their signal peptides promote clamp closing; their mature domain overcomes the rate-limiting ADP release. While repeated ATP cycles shift the motor between unique states, multiple conformationally frustrated prongs in the clamp repeatedly "catch and release" trapped preprotein segments until translocation completion. This universal mechanism allows any preprotein to promiscuously recognize the translocase, usurp its intrinsic dynamics, and become secreted.

SecA: a tale of two protomers.

Bacteria, Archaea and Eukaryotes have evolved a plethora of mechanisms to translocate proteins across their various membranes. The bacterial Sec pathway is ubiquitous and essential for cell viability and is used by most proteins destined for the inner membrane, the periplasm or beyond. In bacteria, Sec system components include the heterotrimers SecY/SecE/SecG and SecD/SecF/YajC and the peripherally associated ATPase motor SecA. SecA in solution is mainly dimeric. Unexpectedly, structures of SecA dimers from different or even the same bacterium do not have a consistent dimerization interface. Analysis of the functional assembled translocase complexes blurs the picture even further as the functional quaternary state of the SecYEG channel is also disputed. Several experimental approaches tried to define the oligomeric state of SecA during preprotein 'pushing' through SecYEG. One high-resolution SecA-SecYEG complex has been visualized. This snapshot might be a step closer to the actual translocating machinery. Nevertheless, because of the use of detergent, the true quartenary state of the translocase might have been disturbed. Hence, even after this and other studies, several issues remain puzzling. New approaches must be combined with current tools to gain insight into the functionally relevant quartenary states of SecA and SecYEG during preprotein translocation.

Long-Lived Folding Intermediates Predominate the Targeting-Competent Secretome.

Secretory preproteins carry signal peptides fused amino-terminally to mature domains. They are post-translationally targeted to cross the plasma membrane in non-folded states with the help of translocases, and fold only at their final destinations. The mechanism of this process of postponed folding is unknown, but is generally attributed to signal peptides and chaperones. We herein demonstrate that, during targeting, most mature domains maintain loosely packed folding intermediates. These largely soluble states are signal peptide independent and essential for translocase recognition. These intermediates are promoted by mature domain features: residue composition, elevated disorder, and reduced hydrophobicity. Consequently, a mature domain folds slower than its cytoplasmic structural homolog. Some mature domains could not evolve stable, loose intermediates, and hence depend on signal peptides for slow folding to the detriment of solubility. These unique features of secretory proteins impact our understanding of protein trafficking, folding, and aggregation, and thus place them in a distinct class.

Preprotein Conformational Dynamics Drive Bivalent Translocase Docking and Secretion.

Most bacterial secretory proteins destined beyond the plasma membrane are secreted post-translationally by the Sec translocase. In the first step of translocation, preproteins are targeted for binding to their 2-site receptor SecA, the peripheral ATPase subunit of the translocase. We now reveal that secretory preproteins use a dual-key mechanism to bridge the signal peptide and mature domain receptor sites and cooperatively enhance their affinities. Docking of targeting-competent mature domains requires that their extensive disorder is finely tuned. This is achieved through amino-terminal mature domain regions acting as conformational rheostats. By being linked to the rheostats, signal peptides regulate long-range preprotein disorder. Concomitant conformational changes in SecA sterically adapt its two receptor sites to optimally recognize hundreds of dissimilar preproteins. This novel intramolecular conformational crosstalk in the preprotein chains and the dynamic interaction with their receptor are mechanistically coupled to preprotein engagement in the translocase and essential for secretion.

Preprotein mature domains contain translocase targeting signals that are essential for secretion.

Secretory proteins are only temporary cytoplasmic residents. They are typically synthesized as preproteins, carrying signal peptides N-terminally fused to their mature domains. In bacteria secretion largely occurs posttranslationally through the membrane-embedded SecA-SecYEG translocase. Upon crossing the plasma membrane, signal peptides are cleaved off and mature domains reach their destinations and fold. Targeting to the translocase is mediated by signal peptides. The role of mature domains in targeting and secretion is unclear. We now reveal that mature domains harbor their own independent targeting signals (mature domain targeting signals [MTSs]). These are multiple, degenerate, interchangeable, linear or 3D hydrophobic stretches that become available because of the unstructured states of targeting-competent preproteins. Their receptor site on the cytoplasmic face of the SecYEG-bound SecA is also of hydrophobic nature and is located adjacent to the signal peptide cleft. Both the preprotein MTSs and their receptor site on SecA are essential for protein secretion. Evidently, mature domains have their own previously unsuspected distinct roles in preprotein targeting and secretion.

Identification of small-molecule inhibitors against SecA by structure-based virtual ligand screening.

The rapid rise of antibiotic-resistant bacteria is one of the major concerns in modern medicine. Therefore, to treat bacterial infections, there is an urgent need for new antibacterials-preferably directed against alternative bacterial targets. One such potential target is the preprotein translocation motor SecA. SecA is a peripheral membrane ATPase and a key component of the Sec secretion pathway, the major route for bacterial protein export across or into the cytoplasmic membrane. As SecA is essential for bacterial viability, ubiquitous and highly conserved in bacteria, but not present in eukaryotic cells, it represents an attractive antibacterial target. Using an in silico approach, we have defined several potentially druggable and conserved pockets on the surface of SecA. We show that three of these potentially druggable sites are important for SecA function. A starting collection of ~500 000 commercially available small-molecules was virtually screened against a predicted druggable pocket in the preprotein-binding domain of Escherichia coli SecA using a multi-step virtual ligand screening protocol. The 1040 top-scoring molecules were tested in vitro for inhibition of the translocation ATPase activity of E. coli SecA. Five inhibitors of the translocation ATPase, and not of basal or membrane ATPase, were identified with IC50 values <65 µm. The most potent inhibitor showed an IC50 of 24 µm. The antimicrobial activity was determined for the five most potent SecA inhibitors. Two compounds were found to possess weak antibacterial activity (IC50 ~198 µm) against E. coli, whereas some compounds showed moderate antibacterial activity (IC50 ~100 µm) against Staphylococcus aureus.